1. Field of the Invention
The present invention relates generally to the field of vaccination, and particularly concerns vaccination to protect animals (e.g., humans) against toxins which induce or cause vascular leak syndrome (VLS) and/or catalyze enzymatic activity that damage or kill living cells. The invention provides immunogens which have been mutated to lack amino acid sequences which induce VLS and other toxic side effects. Disclosed are methods for mutating DNA segments encoding antigens so that an immunogen is produced that lacks sequences that induce VLS and other toxic side effects. The present invention also relates the use of mutated toxins as vaccines to protect immunized individuals from later toxicity due to contact with wild-type toxins.
2. Description of Related Art
Vascular leak syndrome (VLS) is a dangerous effect of various toxins and cytokines in animals involving damage to vascular endothelial cells (ECs) and extravasation of fluids and proteins resulting in interstitial edema, weight gain and, in its most severe form, kidney damage, aphasia, and pulmonary edema (Soler-Rodriguez et al., 1993; Sausville and Vitetta, 1997; Baluna and Vitetta, 1996; Engert et al., 1997). The mechanisms underlying VLS are unclear and are likely to involve a cascade of events which are initiated in endothelial cells (ECs) and involve inflammatory cascades and cytokines (Engert et al., 1997). Proteinaceous toxins have been implicated as contributing to vascular leak syndrome (VLS) (Soler-Rodriguez et al., 1993; Baluna et al., 1996). An amino acid motif (x)D(y) has been identified in various proteinaceous toxins and cytokines that contributes to VLS (Baluna et al., 1999). Mutations in this sequence have been shown to reduce the ability of a peptide to produce the effects associated with VLS (Baluna et al., 1999).
In addition to induction of vascular leak syndrome, proteinaceous toxins often catalyze reactions that are detrimental to living cells. One type of enzymatic toxin is ribosome inactivating proteins (RIPs), which comprise various N-glycosylases. Class one RIPs remove a single adenine from a conserved stem loop rRNA sequence (Endo and Tsurugi, 1998). Class two RIPs contain an N-glycosylase and complex to cell surface binding proteins, which enhances cellular uptake. One of the best studied catalytic toxins is ricin, a class two RIP (Mlsna et al., 1993). One active molecule of the A chain of ricin in the cytosol is sufficient to kill a cell (Musishkin and Wool, 1995). Various residues have been identified whose mutation produces catalytically inactive ricin A chain (Mlsna et al., 1993; Musishkin and Wool, 1995).
Several vaccination attempts have been made to protect individuals from the damage produced by contact with such toxins, including administration of a sub-lethal dose of a toxin (Hewetson et al., 1993), use of a toxoid (Rippy et al., 1991; Hewton et al., 1995) or a sub-unit of a toxin (Lemley et al., 1993). However, despite these attempts, there still remains a need for improved compositions and methods for preparing large amounts of safe and effective proteinaceous toxin vaccines.